Glyphosate tolerant corn event VCO-O1981-5 and kit and method for detecting the same

Abstract

The present invention relates to the field of plant transformation with genes conferring tolerance to glyphosate. The invention particularly relates to a maize (corn) plant transformed with a gene encoding an EPSPS providing the plant tolerance to an application of glyphosate under conditions where this herbicide is effective in killing weeds. The invention particularly concerns an elite transformation event VCO-O1981-5 comprising the gene construct and means, kits and methods for detecting the presence of the said elite event.

Description

The present invention relates to the field of plant transformation with genes conferring tolerance to glyphosate. The invention particularly relates to a maize (corn) plant transformed with a gene encoding an EPSPS providing the plant tolerance to an application of glyphosate under conditions where this herbicide is effective in killing weeds.

The invention particularly concerns an elite transformation event comprising the gene construct and means, kits and methods for detecting the presence of the said elite event.

BACKGROUND OF THE INVENTION

Glyphosate tolerant plants are known in the art and well studied in the past two decades. Glyphosate is an herbicide inhibiting EPSPS which is an enzyme whose activity is upstream of the aromatic amino acids pathway leading to the synthesis of the amino acids tyrosine, tryptophan and phenylalanine. Since glyphosate is a systemic total herbicide, tolerance in the plant when the herbicide is sprayed under usual agronomic conditions may only be achieved by genetic modification of all cells of the plants with an heterologous gene coding for a glyphosate insensitive EPSPS enzyme, either mutated or selected from microorganisms known to have evolved such insensitive EPSPS enzyme.

Glyphosate insensitive EPSPS, gene constructs and plants transformed with said gene constructs are disclosed inter alia in EP 507 698, EP 508 909, U.S. Pat. No. 4,535,060, U.S. Pat. No. 5,436,389, WO 92/04449, WO 92/06201, WO 95/06128, WO 97/04103, WO 2007/064828 and WO 2008/100353, and in references cited herein.

The biophysical characteristics of the EPSPS protein are essential to achieve a good level of tolerance to glyphosate. However, the choice of regulatory elements providing an adequate expression level of the insensitive protein in the plant is also important, as well as the selection of a transformation event, corresponding to a stable line with a stable and limited number of copies of the gene being inserted in the genome of the plant, as well as its stability in the locus where the gene has been inserted is also important to obtain glyphosate tolerance at a commercial level, sufficient for the plant to be used for the preparation of seeds to be planted in a field with a level of tolerance to glyphosate under agronomic conditions sufficient to allow use of the herbicide at effective concentrations to kill the weeds without affecting growing conditions and yields of the crop transformed with the gene encoding EPSPS protein.

Transformation events selected for the preparation of commercial varieties of glyphosate tolerant maize (corn) are known in the art, particularly disclosed in U.S. Pat. No. 6,040,497 and EP 1 167 531.

These varieties of the first generation used for the preparation of commercial plants currently used in the field have some drawbacks.

The event GA21 disclosed in U.S. Pat. No. 6,040,497 comprise multiple copies of a gene construct comprising a rice actin promoter and intron, a sequence coding for an optimized transit peptide, as disclosed in EP 505 909 and a sequence coding for a mutated plant EPSPS comprising two mutations as disclosed in WO 97/04103. The commercially required level of tolerance in the transformation event is obtained with a complex transit peptide and multiple copies of the chimeric gene construct.

The event NK603 disclosed in EP 1 167 531, is also a complex event with the combination of two gene constructs in one locus. The first gene construct comprises a rice actin promoter and intron, with a sequence coding for an Arabidopsis EPSPS transit peptide and a sequence coding for a type II EPSPS resistant to inhibition by glyphosate, isolated from Agrobacterium strain CP4. The second gene construct comprises the CaMV 35S promoter and the rice actin intron, with a sequence coding for an Arabidopsis EPSPS transit peptide and a sequence coding for a type II EPSPS resistant to inhibition by glyphosate, isolated from Agrobacterium strain CP4.

There is a need for a new generation of transformation events allowing a high glyphosate tolerance to maize (corn) plants grown under agronomic conditions with a single copy of the foreign gene construct in the plant genome.

SUMMARY OF THE INVENTION

The invention concerns a maize (corn) plant comprising the event VCO-O1981-5 representative seeds deposited with NCIMB with accession number 41842.

The invention also concerns a maize (corn) plant comprising the VCO-O1981-5 event characterized by the presence of a genomic flanking sequence-gene construct junctions comprising the sequences of SEQ ID NO: 1 and/or SEQ ID NO: 2 or SEQ ID NO: 3.

The invention also concerns corn plants progenies comprising the VCO-O1981-5 event of the invention, characterized by the presence of the said junctions sequences.

Probes to identify the presence of said junction sequences in a maize (corn) plant genome, as well as kits and methods for such identification comprising said probes and their uses, particularly a method for the detection of the VCO-O1981-5 event and primers, probes and a kit for such a detection are also part of the invention.

DETAILED DESCRIPTION OF THE INVENTION

"Transformation event" means a product of plant cell transformation with a heterologous DNA construct, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion of the gene construct into a particular genome location.

"Gene construct" means, according to the invention, a gene constructed from different nucleotide sequences, comprising regulatory elements controlling the expression and translation of a coding sequence in a host organism. The host organism in the invention is particularly maize (corn), cells, tissues and whole plants. The gene construct comprises a promoter region, operably linked to a coding sequence and a terminator region. It may comprise enhancers, such as introns, generally linked downstream the promoter region and upstream the coding region. In the case of glyphosate tolerance, the coding sequence comprise a sequence coding for a chloroplast transit peptide, linked to the sequence coding for an EPSPS enzyme selected for its resistance to inhibition by glyphosate, either mutated or selected or selected and mutated from microorganism having developed resistance to glyphosate.

The gene construct in the event of the invention comprises a DNA molecule of a sugarcane ubiquitin promoter and intron, operably linked to a DNA molecule coding for the maize acetohydroxyacid synthase (AHAS) transit peptide, operably linked to a DNA molecule coding for the Arthrobacter globiformis EPSPS GRG23ACE5. The gene construct also comprises a terminator sequence, particularly the terminator sequence of the 35S CaMV transcript.

The various elements of the gene construct of the invention are isolated and operably linked according to usual techniques of molecular biology known and available to the person skilled in the art.

"Ubiquitin promoter and intron" means the promoter from sugarcane ubiquitin-4 gene and the intron from sugarcane ubiquitin-4 gene, from the non-coding 5' region of the ubiquitin-4 gene of Saccharum officinarum L. as disclosed in Albert and Wei (U.S. Pat. No. 6,638,766) and set forth in SEQ ID NO: 4 and 5, respectively.

"Maize AHAS chloroplast transit peptide" is the N-terminal transit peptide sequence derived from the Zea mays L. (maize) acetohydroxyacid synthase (AHAS) gene, as disclosed in Fang et al (1992) and set forth in SEQ ID NO: 6.

"Arthrobacter globiformis epspsgrg23ace5" means the nucleotide sequence as set forth in SEQ ID NO: 28 of WO 2008/100353. (SEQ ID NO: 7).

"35 CaMV terminator sequence" is the non-coding 3' end from the cauliflower mosaic virus which terminates mRNA transcription and induces polyadenylation as disclosed in Gardner et al (1981) and set forth in SEQ ID NO: 8.

"Plant transformation" and selection of transformed plants is widely disclosed in the art, and more particularly corn transformation. Techniques for corn transformation and breeding are now well known in the art, and particularly disclosed in laboratory notebooks and manuals such as "Transgenic Plants: Methods and Protocols (Methods in Molecular Biology)" (Leandro Pena, Humana Press Inc., 2005), "Heterosis and Hybrid Seed Production in Agronomic Crops" (Amarjit Basra, The Harwoth Press Inc., 1999) and "The Maize Handbook" (Michael Freeling and Virginia Walbot, Springer Lab Manuals, 1994). The transformation of corn is more particularly performed with an Agrobacterium mediated transformation comprising a transformation vector (Hiei and Komari, 1997, U.S. Pat. No. 5,591,616).

The transformation of a plant with a gene construct generally comprises the steps of a) inoculating a plant cell with a strain of Agrobacterium tumefaciens comprising a transformation vector comprising the gene construct; b) selecting the plant cells having integrated into their genome the gene construct of the invention; c) regenerating a fertile plant from the selected plant cell; d) pollinating the regenerated plant, and; e) selecting progeny plants tolerant to high doses of glyphosate, then; f) selecting the plants having stably integrated one unique copy of the gene construct of the invention.

"Transformation vectors" means a DNA molecule comprising the gene construct and additional DNA elements allowing introduction of the gene construct into a plant cell and integration of said gene construct into the genome of the plant cell. Transformation is an Agrobacterium mediated transformation, wherein the transformation vector comprises right and left borders of a T-DNA plasmid from Agrobacterium tumefaciens flanking the gene construct to be inserted. Such transformation vectors are well disclosed in the art and readily available to the person skilled in the art of plant molecular and cellular biology and plant transformation.

"Right and left borders of a T-DNA plasmid from Agrobacterium tumefaciens" are DNA sequences of the right and left border sequences from Ti plasmids and well known and disclosed in the art of plant transformation. More particularly, the right border (RB) sequence is used as the initiation point of T-DNA transfer from Agrobacterium tumefaciens to the plant genome, it is particularly the right border sequence of nopaline type T-DNA derived from plasmid pTiT37. (Depicker et al. 1982; Komari et al., 1996). The left border (LB) sequence defines the termination point of T-DNA transfer from A. tumefaciens to the plant genome, it is particularly the left border sequence from Ti plasmid pTiC58. (Komari et al., 1996; Otten et al., 1999).

"Transformed plants" mean plants having integrated into their genome the gene construct flanked with the full or a fragment of the sequence of the right and left borders of a T-DNA plasmid from Agrobacterium tumefaciens. All cells of the transformed plants have integrated into their genome the gene construct. The transformed plant is a fertile plant and more particularly a plant which agronomic properties (yield, grain quality, drought tolerance, etc.) are not impaired compared to the same plant not transformed.

"Insert DNA" is the gene construct flanked by RB and LB sequences and inserted in the plant genome at a specific locus.

The event is defined by a stable integration of the insert T-DNA of the invention at a specific locus in the maize (corn) genome.

The insertion defines two unique junctions DNA sequence wherein the insert T-DNA sequence joins the flanking maize genomic sequences. By reference to the insert T-DNA, there is a 5' junction DNA localized in the 5' part of the insert T-DNA and a 3' junction DNA localized in the 3' part of the insert T-DNA. Non limiting examples of the event VCO-O1981-5 junctions DNA (or so called "event VCO-O1981-5 DNA") are set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 3.

The term "event" refers to the original transformed plant and progeny of the transformed plant that include the heterologous DNA. The term "event" also refers to progeny produced by a sexual outcross between the transformed plant and another variety in that the progeny includes the heterologous DNA.

The term event also refers to progeny produced by sexual backcrosses between a donor inbred line (the original transformed line and the progeny) comprising the insert DNA and the adjacent flanking genomic sequences and a recipient inbred line (or recurrent line) that does not contains the said insert DNA. After repeated back-crossing, the insert DNA is present in the recipient line at the same locus in the genome as in the donor line.

The term "event" or event sequence of VCO-O1981-5 also refers to the insert DNA from the original transformed plant comprising part or all of the insert DNA and adjacent flanking genomic sequences that would be transferred from the donor line to the recipient line.

The last backcross progeny would be selfed to produce progeny which are homozygous for the introgressed insert DNA.

These progeny would then be used as inbred parent line to produce hybrids.

A glyphosate tolerant maize (corn) VCO-O1981-5 (also named 6981 maize (corn)) can be bred by first sexually crossing a donor parental maize (corn) plant consisting of a maize (corn) plant grown from the transgenic maize (corn) plant VCO-O1981-5 (also named 6981 maize (corn)); representative seeds deposited with NCIMB with accession number 41842 and progeny thereof derived from transformation with the expression cassettes of the present invention that tolerates application of glyphosate herbicide, and a recipient parental maize (corn) plant that lacks the tolerance to glyphosate herbicide, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is tolerant to application of glyphosate herbicide; and selfing the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from the second progeny plants a glyphosate herbicide tolerant plant. These steps can further include the back-crossing of the first glyphosate tolerant progeny plant or the second glyphosate tolerant progeny plant to the recipient parental (or recurrent) maize (corn) plant or a third parental maize (corn) plant, thereby producing a maize (corn) plant that tolerates the application of glyphosate herbicide.

Methods for producing a hybrid maize (corn) seed are well known in the art. The method comprises crossing the plant comprising the VCO-O1981-5 event deposited on 13 May 2011 by GEMSTAR, rue Limagrain, BP-1, 63720 Chappes, FRANCE, with NCIMB with accession number 41842 or said plant progeny comprising the VCO-O1981-5 event with a different maize (corn) plant and harvesting the resultant hybrid maize (corn) seed comprising the VCO-O1981-5 event.

It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two or more independently segregating added, transgenes. A method for producing a maize (corn) plant that contains in its genetic material two or more transgenes, wherein the method comprises crossing the maize (corn) plant comprising the VCO-O1981-5 event deposited with NCIMB with accession number 41842 or said plant progeny comprising the VCO-O1981-5 event with a second plant of maize (corn) which contains at least one transgene so that the genetic material of the progeny that results from the cross contains the transgene(s) operably linked to a regulatory element and wherein the transgene is selected from the group consisting of male sterility, male fertility, insect resistance, disease resistance and water stress tolerance and herbicide resistance (wherein the transgene confers resistance to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate).

Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes. Said maize (corn) plant comprising two or more transgenes would be used to produce hybrid maize (corn) seeds wherein the method comprises crossing the said maize (corn) plant with a different maize (corn) plant and harvesting the resultant hybrid maize (corn) seeds comprising two or more transgenes.

Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several references, e.g., A. Hallauer and J. B. Miranda in Quantitative genetics in maize breeding. (2nd edition, Iowa State University press) and R. Bernardo in Breeding for quantitative traits in plants. (Stemma press.com).

The term event also refers to a maize (corn) plant produced by vegetative reproduction from the maize (corn) plant comprising the VCO-O1981-5 event deposited with NCIMB with accession number 41842 or said plant progeny comprising the VCO-O1981-5 event. Vegetative reproduction can be initiated from a plant part as for example cells, tissues such as leaves, pollen, embryos, roots, root tips, anthers, silks, flowers, kernels, ears, cobs, husks, stalks or tissue culture initiated from said plant part. The term event also refers to said plant part.

The term event concerns a glyphosate tolerant corn, comprising in its genome the nucleotide sequences that are at least 95%, preferably at least 96, 97, 98, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

The invention also concerns the polynucleotide sequences comprising SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 and having any length from 25 nucleotides to 5092 nucleotides.

Particularly the invention concerns the polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 specific to event VCO-O1981-5. These polynucleotide sequences are suitable for selectively identifying the event VCO-O1981-5 in different biological samples. By biological samples, it is to be understood a plant, plant part or plant material such as cells, tissues as leaves, pollen, embryos, roots, root tips, anthers, silks, flowers, kernels, ears, cobs, husks, stalks or seeds. It is also to be understood a processed products comprising or derived from plant part or plant material.

Methods for the detection of the presence or absence of specific DNA elements in a plant genome are well known in the art. Main techniques comprise DNA sequence amplification, particularly with Polymerase Chain Reaction, with specific primers allowing amplification of the DNA sequence, and hybridization with a probe specific for the DNA sequence.

The invention comprises a method for the identification of the presence or the absence of the transformation event VCO-O1981-5 of the invention, particularly with one of the known techniques.

In a particular embodiment of the invention, the method comprises the steps of: a) extracting DNA from a biological sample obtained from a maize (corn) plant, tissue or cell; b) contacting said extracted DNA with a first and second primers of appropriate length selected to allow production of an amplicon DNA molecule comprising all or part of the event sequence of VCO-O1981-5; c) performing an amplification reaction to produce amplicon DNA molecules, and; d) detecting the presence or the absence of a nucleotide sequence comprising all or part of the event sequence of VCO-O1981-5 in the amplicon molecule.

Primers have generally a length comprised between 10 and 30 nucleotides, and are selected and prepared according to techniques well known to the person skilled in the art of molecular biology.

In a particular embodiment of the invention, the amplicon molecule comprising all or part of the event sequence of VCO-O1981-5 comprises the event junction sequence set forth in SEQ ID NO: 1 and/or the event junction sequence set forth in SEQ ID NO: 2 and/or a sequence that is at least 95%, preferably at least 96, 97, 98, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

Advantageously, the first and second primers comprises sequences homologous to a sequence fragment of the event sequence set forth in SEQ ID NO: 3, and are selected to be flanking the event VCO-O1981-5 sequence and to generate an amplicon comprising the DNA sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2.

Preferred primers comprise the DNA sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12.

In another embodiment of the invention, the method comprises the steps of: a) extracting DNA from a biological sample obtained from a maize (corn) plant, tissue or cell; b) contacting said extracted DNA with probe(s) of sufficient length to hybridize under stringent conditions with a nucleotide sequence that specifically detect at least one of VCO-O1981-5 junction sequence; c) subjecting the extracted DNA and probe(s) to stringent hybridization conditions, and; d) detecting the hybridization of the probe(s) to the extracted DNA, wherein detection indicates the presence of an event VCO-O1981-5 sequence.

The invention also concerns a method for producing a glyphosate tolerant plant comprising breeding a plant of the invention, comprising the event VCO-O1981-5 sequence, and selecting progenies by detecting the presence of the event VCO-O1981-5 sequence, particularly with the detection method of the invention.

"Amplicon" refers to the product obtained by amplification with a specific pair of primers of a target nucleotide sequence comprised in a nucleotide template sequence.

Primers, probes and methods for the identification of the presence or absence of a specific DNA or amplicon sequence in a corn genome are well known in the art, particularly disclosed in paragraphs [0027] to [0043] of EP 1 167 531 which are incorporated herein by reference, as well as publications cited herein.

Stringent conditions are defined as following. For sequences comprising more than 30 bases, Tm is defined by the equation: Tm=81.5+0.41 (% G+C)+16.6 Log (concentration in cations)-0.63 (% formamide)-(600/number of bases) (Sambrook et al., 1989).

For sequences shorter than 30 bases, Tm is defined by the equation: Tm=4(G+C)+2(A+T).

Under appropriate stringency conditions, in which non-specific (aspecific) sequences do not hybridize, the temperature of hybridization is approximately between 5 and 30.degree. C., preferably between 5 and 10.degree. C. below Tm and hybridization buffers used are preferably solutions of higher ionic force like a solution 6*SSC for example.

The invention also concerns a kit for detecting the presence or absence of the VCO-O1981-5 event of the invention in a biological sample, wherein it comprises primers and/or probes amplifying or hybridizing to a polynucleotide sequence comprising an event VCO-O1981-5 DNA sequence.

The invention particularly comprises a first primer of 10 to 30 nucleotides, comprising a sequence homologous to a sequence fragment of SEQ ID NO: 3 and a second primer of 10 to 30 nucleotides comprising a sequence having complementarity to a sequence fragment of SEQ ID NO: 3, the first and the second primers flanking an event VCO-O1981-5 DNA sequence and generating an amplicon molecule comprising SEQ ID NO: 1 or SEQ ID NO: 2.

Particularly, said first and second primers comprise the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The invention also concerns an isolated nucleotide sequence comprising, or consisting essentially of, a sequence set forth in SEQ ID NO: 11 and/or SEQ ID NO: 12.

The invention also concerns an isolated nucleotide sequence comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2, particularly comprising, or consisting essentially of, the sequence set forth in SEQ ID NO: 3 or a fragment thereof and/or a sequence that is at least 95%, preferably at least 96, 97, 98, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

Techniques for gene constructions as well as techniques for gene identification using amplification techniques such as PCR or hybridization techniques are well known in the art, and particularly disclosed in laboratory notebooks and manuals such as Sambrook & Russel (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

FIGURES

FIG. 1 represents the transformation vector pAG3541.

FIG. 2 represents the schematic diagram of the selection of event VCO-O1981-5.

FIG. 3 describes the breeding diagram for event VCO-O1981-5.

FIG. 4 represents the EPSPS GRG23ACE5 expression cassette within the T-DNA region.

FIG. 5 represents a segregation analysis carried out in the following generation for the B110 and B109 crosses, and in the next 4 generations for line AAX3.

EXAMPLES

Abbreviations, Acronyms, and Definitions

TABLE-US-00001 AHAS Acetohydroxyacid synthase BLAST Basic Local Alignment Search Tool Bp Base pair CaMV Cauliflower mosaic virus CHI-test Pearson's chi-square test CTP Chloroplast transit peptide DNA Deoxyribonucleic acid EPSPS 5-enolpyruvylshikimate-3-phosphate synthase (protein) epsps 5-enolpyruvylshikimate-3-phosphate synthase (DNA sequence) FST Flanking sequence tag GRG23ACE5 Modified EPSPS from Arthrobacter globiformis kbp kilobase pairs LB Left border PCR Polymerase chain reaction RB Right border T-DNA Transferred-DNA Ti Tumor-inducing Vir Virulence genes of Agrobacterium

I. Production of Glyphosate Tolerant Event VCO-O1981-5

Maize event VCO-O1981-5 was generated using a standard Agrobacterium mediated transformation protocol (Hiei and Komari, 1997). Agrobacterium contains a tumour-inducing (Ti) plasmid, which includes virulence (vir) genes and a transferred-DNA (T-DNA) region. Genes of interest can be inserted into the T-DNA region and thereafter transferred to the plant nuclear genome. The use of a Ti plasmid with the tumor-inducing genes deleted is commonly known as disarmed Agrobacterium-mediated plant transformation. Wounded plant cells produce phenolic defense compounds, which trigger the expression of the Agrobacterium vir genes. The encoded virulence (Vir) proteins process the T-DNA region from the Ti-plasmid, producing a `T-strand`. After the bacterium attaches to a plant cell, the T-strand and several types of Vir proteins are transferred to the plant through a transport channel. Inside the plant cell, the Vir proteins interact with the T-strand, forming a T-complex. This complex targets the nucleus, allowing the T-DNA to integrate into the plant genome and express the encoded genes (Gelvin, 2005).

The recipient organism is the dent type of Zea mays, which belongs to the genus Zea of the family Gramineae (Hi-II stock material). This material is supplied in the form of two separate lines Hi-IIA and Hi-IIB. These lines are then crossed and the resulting embryos are used as target tissue for transformation. Hi-IIA and Hi-IIB are partially inbred lines selected out of a cross between corn inbred lines A188 and B73. As the recipient organism, hybrid Hi-II of Zea mays was produced by crossing the partially inbred Hi-IIA and Hi-IIB lines which were obtained from Maize Genetics COOP Stock Center (Urbana, Ill., USA). The T-DNA region in transformation vector pAG3541 was introduced using Agrobacterium into the hybrid Hi-II by co-cultivation (approximately 72 hours at 22.degree. C. in the dark) with immature maize embryos. Transformed callus was selected on glyphosate-containing medium as a selective agent. The antibiotic timentin (200 ppm) was included in tissue culture media to eliminate Agrobacterium cells from the callus after transformation (Cheng et al., 1998).

The transformation vector pAG3541 (FIG. 1) was used to transfer the epsps grg23ace5 expression cassette to maize. Only the T-DNA existing between the right and left border (RB and LB) sequences respectively is integrated into the maize genome. The DNA regions outside the T-DNA borders are not transferred. Outside these borders bacterial antibiotic resistant marker genes are required for the introduction and maintaining of the vector in the Agrobacterium cells. The vir genes are required for the production of the T-DNA transfer complex (De la Riva et al., 1998).

Out of 100 events generated in T0, VCO-O1981-5 event was selected through multiple evaluation field trials for glyphosate tolerance and agronomic performances like germination, vegetative characteristics (such as plant height, grain weight) and reproductive characteristics (such as days to 50% pollen shed, days to 50% silking, yield).

The schematic diagram of the selection of event VCO-O1981-5 is provided on FIG. 2.

Event VCO-O1981-5 was also selected for good molecular characteristics based on the unicity and integrity of the insert and the stability of the genomic insertion locus and its inheritance.

More specifically, event VCO-O1981-5 was selected for its low level of allergenicity risk. Twelve Open Reading Frames (ORFs), created by the insertion of the T-DNA in the genome, have been identified at the junctions between the T-DNA and the maize genome. For this analysis, we consider that ORFs are any potential coding region between two stop codons as defined by the European Food safety Authority (EFSA). Bioanalysis of ORFs was first performed, followed by analysis for putative allergenic motifs in the determined ORFs using an 80 amino acids (AA) sliding window and 8 AA exact match. Analysis was performed according to Codex Alimentarius (2003) and using AllergenOnline Database Version 11 from February 2011 (http://www.allergenonline.org/databasefasta.shtml). Two potential hits were identified using the 80 AA sliding window, but it is highly unlikely that the identified genetic sequence would generate a translatable mRNA sequence and since these sequences were identified from the native maize genome, there is no impact to the allergenicity risk assessment. Finally, event VCO-O1981-5 was selected due to its advantageous location in a genomic region harboring a good recombination rate. This characteristic is notably important for the conversion program in which the event will be further used.

FIG. 3 describes the breeding diagram for event VCO-O1981-5.

II. Donor Genes and Regulatory Sequences

A. Transformation Vector Map

Event VCO-O1981-5 was produced by disarmed Agrobacterium-mediated transformation using the plasmid pAG3541. This transformation vector contains the epsps grg23ace5 expression cassette within the T-DNA region (FIG. 4).

B. Description of the Genes and Regulatory Sequences

A synthetic coding region sequence comprising a maize chloroplast transit peptide (acetohydoxyacid synthase) (Fang et al., 1992) and a gene encoding EPSPS GRG23ACE5 enzyme was generated. The synthetic gene was subcloned downstream from the ubiquitin-4 promoter from Saccharum officinarum L. (Albert and Wei, 2003) and upstream from the terminator 35S of Cauliflower mosaic virus (Gardner et. al., 1981) to create plasmid pAX3541. The promoter:gene::terminator fragment from this intermediate plasmid (based on pSB11, Japan Tobacco, Inc. (Hiei and Komari, 1997)) was mobilized into Agrobacterium tumefaciens strain LBA4404, which also harbors the plasmid pSB1, using triparental mating and plating on media containing spectinomycin, streptomycin, tetracycline and rifampicin to form a final plasmid, pAG3541. Rifampicin is included as an additional selection for Agrobacterium as the rifampicin resistance marker gene is present in the Agrobacterium chromosomal DNA. The integrity of cointegrate product of pSB1 and pAX3541-plasmid pAG3541 was verified by Southern hybridization.

The amino acid sequence of the wild-type EPSPS isolated from Arthrobacter globiformis was altered using a directed evolution technique resulting in the EPSPS GRG23ACE5 protein described herein and expressed in event VCO-O1981-5. The deduced amino acid sequence of the EPSPS GRG23ACE5 protein is shown below (SEQ ID NO: 23).

TABLE-US-00002 metdrlvipg sksitnrall laaaakgtsv lvrplvsadt safktaiqal ganvsadgdd wvveglgqap nldadiwced agtvarflpp fvaagqgkft vdgseqlrrr plrpvvdgir hlgarvsseq lpltieasgl aggeyeieah qssqfasgli maapyarqgl rvkipnpvsq pyltmtlrmm rdfgietstd gatvsvppgr ytarryeiep dastasyfaa asavsgrrfe fqglgtdsiq gdtsffnvlg rlgaevhwas nsvtirgper ltgdievdmg eisdtfmtla aiapladgpi titnigharl kesdrisame snlrtlgvqt dvghdwmriy pstphggrvn chrdhriama fsilglrvdg itlddpqcvg ktfpgffdyl grlfpekalt lpg

III. Transgene Copy Number Analysis

Maize genomic DNA was isolated (Dellaporta et al., 1983) and quantified by fluorimetry. DNA restriction, gel electrophoresis, Southern blotting and hybridization with radiolabeled probes were carried out according to standard procedures (Sambrook et al., 1989). Total genomic DNA was purified from event VCO-O1981-5 and digested with appropriate restriction endonucleases to determine both insert copy number and insert integrity.

Templates for radioactive probes synthesis were prepared using standard PCR methods. Oligonucleotide primers specific to promoter and terminator sequences in the T-DNA were used to generate a DNA probe specific for the T-DNA insert. The DNA probe was labeled with .sup.32P .alpha.-dCTP using Ready-To-Go DNA labeling beads (GE Health). The labeled probe was purified over Micro Bio-Spin P-30 Tris-Chromatography Columns (BioRad). Hybridizations were carried out at 65.degree. C. (Church, 1984). After hybridization, blots were washed at 65.degree. C., with the final wash containing 1% (w/v) sodium dodecyl sulfate at pH 7.0. Blots were exposed to Kodak AR X-OMAT film using a Kodak intensifying screen at -80.degree. C.

Genomic DNA from event VCO-O1981-5 corn, BC1 negative segregant corn, and B110 inbred corn was digested with the restriction enzymes HindIII and NdeI (New England Biolabs, Ipswich, Mass.) independently. Each of these restriction enzymes cuts once within the T-DNA region. When hybridized with the epsps grg23ace5 gene probe, the resulting number of hybridization products would indicate the insert copy number within the maize genome. Both digests produced a single band indicating a single copy of the insert present.

Genomic DNA from event VCO-O1981-5 corn, BC1 negative segregant corn, and B110 inbred corn was digested with a combination of HindIII and EcoRI, and independently with MfeI (New England Biolabs, Ipswich, Mass.). A set of four independent probes (ScUbi4 promoter, ScUbi4 intron, epsps grg23ace5 gene, and 35S terminator) were used to confirm the integrity of the expression cassette structure The results of the analysis indicated that the epsps grg23ace5 expression cassette was intact and the functional components were found and verified in the expected order in the inserted DNA.

Southern blot analysis was conducted to verify the absence of the transformation plasmid components outside of the transferred T-DNA region. Maize genomic DNA (VCO-O1981-5 event and appropriate negative controls) was digested with a combination of HindIII and EcoRI, and independently with MfeI (New England Biolabs, Ipswich, Mass.). The Agrobacterium plasmid pAG3541 was included as a positive control for hybridization of the transformation plasmid components. The probes used were designed to hybridize to the functional components of the plasmid including the sequence of aad, tetR, tetA, oriT, virC, virG, and virB.

Southern blot analysis results indicate that none of the vector probes hybridized to VCO-O1981-5 genomic DNA confirming the absence of the sequences of the functional components of the plasmid in event VCO-O1981-5. These same probes however did show hybridization with the plasmid vector control on each blot indicating that if the vector sequences were inadvertently transferred to event VCO-O1981-5 corn, they would have been detected in this analysis.

Southern blot analysis was conducted on multiple generations of event VCO-O1981-5 progeny to evaluate the stability of the T-DNA sequence insertion. Genomic DNA isolated from leaf material of VCO-O1981-5 plants from four successive breeding generations (BC0, BC1, BC3, and BC4) and negative controls were digested with the restriction enzyme HindIII (New England Biolabs, Ipswich, Mass.) which, as noted earlier, cuts once within the T-DNA region. When hybridized with the probe specific for the epsps grg23ace5 gene, VCO-O1981-5 produces a single band approximately 4.0 kb in size. The transformation plasmid pAG3541 was included as a hybridization control. All four generations analyzed showed an identical hybridization pattern producing the identical 4.0 kb band. If the genetic insert were unstable within the maize genome through successive breeding of the event, one would expect to detect changes in the banding pattern produced. The data indicates a stable insertion site in event VCO-O1981-5.

IV. Sequencing of the Insert and Flanking Genomic DNA

Southern blot analysis has demonstrated that event VCO-O1981-5 contains a single intact T-DNA insert containing a single expression cassette. The sequence of the transgenic locus including 5' and 3' FSTs (flanking sequence tags) and the sequence of the pre-insertion locus (locus in the corn genome where the transgene was inserted) have been determined.

The maize genomic sequences flanking the T-DNA insertion in event VCO-O1981-5 were obtained by Genome Walker.TM. (Clontech) (5'FST) and direct PCR (3'FST). Using the DNA sequences generated, a BLAST search (Altschul et al., 1997) was performed against the Maize Genetics and Genomics Database (Lawrence et al., 2004). Both the 5' and 3' FST sequences mapped to chromosome 1.

700 bp were obtained for the 5' FST and 700 bp for the 3' FST. The enzyme SspI was used for generating the library. The T-DNA specific primers used are listed in the following Table 1.

TABLE-US-00003 TABLE 1 SEQ Primer 5'-->3' sequence ID 3'FST Ace5-1 ACAGGATCGCTATGGCGTTTTCAATCC 17 Ace5-2 ATGCGTCGGGAAGACCTTTCCTGGCTTC 18 O39 CACCAGGGAGGAGGCAACAACAAGTAG 19 5'FST Scubi-NewR AGAAAGAGTCCCGTGAGGCTACGGCAC 20 Scubi2-Rev CTGGGATTTGGATGGATGAGGCAAGGAG 21 Scubi1-Rev AGAGGTCGCCGCGGAGATATCGAGGAG 22

The insertion site could be mapped using a BLAST search against the Maize Genetics and Genomics Database (http://www.maizegdb.org/). It is located in the chromosome 1, more precisely on the BAC: AC185611.

To confirm the FST result, primers were deduced from the sequence obtained by the Genome Walker strategy and used to directly amplify the 5' and 3' FST sequence from Hi-II and VCO-O1981-5 (6981). The expected PCR products were obtained and sequenced. The sequences obtained were found identical as the one obtained from the Genome Walker which is thus considered as accurate.

The Map of inserted T-DNA, gene construct of the invention flanked with the right and left border and the flanking sequences (SEQ ID NO: 9 and SEQ ID NO: 10) is described on FIG. 4.

The 3' flanking sequence (SEQ ID NO: 9) has the following sequence:

TABLE-US-00004 gttctcagagggagatgggcggcaagggcggcgggggtggtggcaagggc ggcggcgggggtggtggcaagggcggaggaggttttggtggcaagagcgg cggcgggggtggtggcaagggcggaggaggtgttggtggcaagagcggcg gcggcaagtcaggcggcggcggcggtgggggctatggtggtggagggaag tcaggctccggcggcagtggcggcgacggaatgatgaaggcgcccggcgg cagtggcgagtacatctcccgctctgtcttcgaggccagcccgcaggtgt tcttccatggcctccaccagggaggaggcaacaacaagtagatccatcta gctagactgctgctgctacttcacaagcttgggacgatgtgtgatcatgc atgcttggactggcatcagtctctatgtagcttctgaataaaataaaatg taacgatgctcgattgtgtttcacttgctcgcttgtttcagccaagttat tatatatcatcaggctcgtacgtcagctatatatatatatatatatatat atatatatatatatatatatatatatatatatatatatatatatatatat atatatatatatatacacacacacacatatgcaggtgcatggattgtgca acgcgaatgtgtgattgtgctaatccgttagttgatgccgtttgttgctt

The 5' flanking sequence (SEQ ID NO: 10) has the following sequence:

TABLE-US-00005 tttcctcattttctttttcccgcttttgtttcaatttttcttgggtaatg tacagtgagtatattttttcttgttctttttctcatggccaaaatccaca atggatcgatgaattagctgtcgttgttgccaacaacaacaacagaacaa aatcacgtgacgtactagcacaatgcaagtagccaaactgagcttccggg caccgacgaacggttgcacgccatcggcgggaaggaacaggccgggctgt caatggacaaacgggccgccaagctggagggagtgtcatgggctttgaga accatcgtcagggtccagtttattcttttgtttttattaaaggcggtaaa ctcggggaacgaatatactaggaaaaacactagccagtcagagtcagtca aagtggactgagttaaaattgcaacgacacacacgcagcagtcagggcgt cgggaatgaacaatggatgaatttattataatctgaagaaaacgaaggga cacagccactacgaacactggggagtggggagtgaatgaatgaatgcatt ccactggaccgttccagcgcttcgtgtgcctcgctagatgcgctgaacac tcgaacgccatggacctcgctccgctctctatatatagagggaaggcctt cagtctactcctcgggatataccactgaacgtcaccaagaagatcagtac

Additionally, the entire T-DNA insert in event VCO-O1981-5 was sequenced and verified to be identical to that in transformation vector pAG3541. During the transformation integration process, the right and left border sequences do not typically remain intact and minor deletions in both were identified in event VCO-O1981-5.

A complete sequence comprising the entire T-DNA insert sequence and the flanking genomic sequence is listed as SEQ ID NO: 3.

V. Inheritance of the Glyphosate Tolerant Trait

During performance evaluation of event VCO-O1981-5, the locus containing epsps grg23ace5 was crossed with 3 inbred lines (B110, B109, AAX3). Progeny plants for each line were then sprayed with glyphosate to identify plants that inherited and expressed epsps grg23ace5 and assess the segregation ratio into each of the lines. The progenitor line for testing was generated by pollinating line B110 with the parental T0 plant for event VCO-O1981-5, which yielded a BC0 line (B110.times.VCO-O1981-5). These BC0 seeds were germinated and plants were crossed simultaneously with lines B110, B109 and AAX3. Segregation analysis was carried out in the following generation for the B110 and B109 crosses, and in the next 4 generations for line AAX3 (FIG. 5).

All glyphosate sprays were carried out at either 1.times., 4.times., or 8.times. the spray rate in outdoor field plots (1.times. was 540 g of glyphosate, acid form/ha). Positive segregants that survived the spray were scored as "tolerant", while negative segregants did not survive the spray and were scored as "sensitive".

TABLE-US-00006 TABLE 2 CHI Generation Gly. Obs. Obs. Exp. Exp. test (line) No S.R. Tol. Sens. Tol. Sens. % Tol. value BC1 9 4x 7 2 4.5 4.5 77.8% 0.096 (B110) BC1 7 8x 2 5 3.5 3.5 28.6% 0.257 (B110) BC0 10 4x 5 5 5 5 50.0% 1.000 (B109) BC0 11 8x 5 6 5.5 5.5 45.5% 0.763 (B109) BC0 28 1x 12 16 14 14 42.9% 0.450 (AAX3) BC1 227 1x 100 127 113.5 113.5 44.1% 0.073 (AAX3) BC2 58 1x 29 29 29 29 50.0% 1.000 (AAX3) BC3 74 1x 38 36 37 37 51.4% 0.816 (AAX3) Abbreviations: Gen.: Generation; No: Number of plants; Gly. S.R.: glyphosate spray rate; Obs. Tol: observed tolerant; Obs. Sens.: observed sensitive; Exp. Tol.: expected tolerant; Obs. Tol: expected sensitive; % Tol.: % Tolerant.

All plants were evaluated two weeks after spraying. A segregation ratio of 1:1 was expected in each generation because epsps grg23ace5 is present at single and hemizygous copy in the donor parental line crossed with the lines B109, B110 or AAX3.

Observed segregation patterns were compared to the expected patterns and these data were compared using a chi-squared (X.sup.2) distribution analysis, as follows:

X.sup.2=.SIGMA.[(|o-e|).sup.2/e], where o=observed frequency of tolerance, and e=expected frequency of tolerance.

A chi-square value of .gtoreq.0.05 was treated as the cutoff for statistical support of a 1:1 segregation in each generation, and this value was exceeded for each of the segregation analysis groups. The results of this analysis are consistent with the inheritance of a single copy of epsps grg23ace5 into each of the inbred lines tested (B110, B109, AAX3).

Transformation event VCO-O1981-5 contains a single genetic insertion of the epsps grg23ace5 gene, and that gene is inherited through successive breeding generations in the predictable Mendelian fashion.

VI. Method of Detection of the VCO-O1981-5 Event:

This example describes an event-specific real-time quantitative TaqMan PCR method for determination of the relative content of event VCO-O1981-5 DNA to total maize (Zea mays) DNA in a biological sample.

The PCR assay has been optimized for use in an ABI Prism.RTM. 7900 sequence detection system.

For specific detection of event VCO-O1981-5 genomic DNA, a 85-bp fragment of the region that spans the 5' TDNA insert and flanking genomic junction in maize event VCO-O1981-5, is amplified using two specific primers. PCR products are measured during each cycle (real-time) by means of a target-specific oligonucleotide probe labelled with a fluorescent dye: FAM as a reporter dye at its 5' end and MGBmolecule as a quencher at its 3' end. The 5'-nuclease activity of the Taq DNA polymerase is exploited, which results in the specific cleavage of the probe, leading to increased fluorescence, which is then monitored. For relative quantification of event VCO-O1981-5 DNA, a maize specific reference system amplifies a 70-bp fragment of aldolase (Kelley et al., 1986), a maize endogenous sequence, using a pair of aldolase gene-specific primers and an aldolase gene-specific probe labelled with VIC and TAMRA.

Two types of quantification are simultaneously performed in this method: one for the endogenous gene aldolase and one for the event VCO-O1981-DNA region. The following sets of primers and probes are used.

TABLE-US-00007 TABLE 3 Sequence (5' to 3') VCO-O1981-5 primer F Ccactgaacgtcaccaagaaga (SEQ ID NO: 11) VCO-O1981-5 primer R Gccgctactcgagggattta (SEQ ID NO: 12) VCO-O1981-5 probe 6-FAM-cagtactcaaacactgatag- MGB (SEQ ID NO: 13) Aldolase primer F Agggaggacgcctccct (SEQ ID NO: 14) Aldolase primer R Accctgtaccagaagaccaagg (SEQ ID NO: 15) Aldolase probe 6-VIC-tgaggacatcaacaaaagg cttgcca-TAMRA (SEQ ID NO: 16)

The master-mix for the aldolase reference gene system is prepared as followed in Table 4:

TABLE-US-00008 TABLE 4 Final concentration in Component PCR .mu.l/reaction TaqMan .RTM. Universal Master Mix 2X 1x 12.5 Primer F (5 .mu.M) 300 nM 1.5 Primer R (5 .mu.M) 300 nM 1.5 Probe (5 .mu.M) 200 nM 1.0 Nuclease free water # 6.0 Template DNA (maximum 200 ng) # 2.5 Total volume: 25 .mu.l

The master-mix for VCO-O1981-5 event is prepared as followed in Table 5:

TABLE-US-00009 TABLE 5 Final concentration in Component PCR .mu.l/reaction TaqMan .RTM. Universal Master Mix 2X 1x 12.5 Primer F (5 .mu.M) 300 nM 1.5 Primer R (5 .mu.M) 300 nM 1.5 Probe (5 .mu.M) 200 nM 1.0 Nuclease free water # 6.0 Template DNA (maximum 200 ng) # 2.5 Total volume: 25 .mu.l

Run the PCR with cycling conditions listed below for both VCO-O1981-5 event and aldolase assays in the Applied Biosystems 7900 system.

TABLE-US-00010 TABLE 6 Data Step Stage T .degree. C. Time (sec) collection Cycles 1 Uracil-DNA-N Glycosylase (UNG) 50.degree. C. 120'' no 1x 2 Initial denaturation 95.degree. C. 600'' no 1x 3 Amplification Denaturation 95.degree. C. 15'' no 40x Annealing & 60.degree. C. 60'' yes Extension

VII. Evaluation of Agronomic Performance of Event VCO-O1981-5

In order to evaluate agronomic performance characteristics of event VCO-O1981-5 as compared to an appropriate negative isoline, two experimental varieties were produced and seed used for multiple location evaluation. The experimental varieties are hybrid maize obtained by crossing the event VCO-O1981-5 (BC2S2) with two different lines (B116 and CH01). Negative segregants crossed with the lines B116 and CH01 were used as comparators (see table 5 and FIG. 3 for breeding diagram).

TABLE-US-00011 TABLE 7 Maize hybrids tested in agronomic evaluations. Line Tested Pedigree VCO-O1981-5 (A) BC0S2 VCO-O1981-5 .times. B116 Control: Negative isoline (A) BC0S2 null .times. B116 VCO-O1981-5 (B) BC0S2 VCO-O1981-5 .times. CH01 Control: Negative isoline (B) BC0S2 null .times. CH01

These hybrids were characterized under diverse environmental and growing conditions similar to those used in maize production. The study was conducted using a Randomized Complete Block design with three replications (plots) of each entry per location. Each plot consisted of four, 30-inch rows by 17.5 to 20 ft. long. Plants were thinned prior to reaching the V8 leaf stage resulting in a uniform number of plants in each row. Weeds outside of the plots (in alleyways and borders) managed as to not confound measures of agronomic characteristics. Weeds within the plots were managed by conventional herbicides and cultural practices (hand hoeing). No broad spectrum herbicides were applied to the study or borders rows except as a pre-plant or pre-emergence application. Data on all traits was collected on the middle two rows of each four row plot. Data collected over season is summarized in Tables 8 and 9.

TABLE-US-00012 TABLE 8 Agronomic performance results-vegetative characteristics Agronomic VCO- Number Number Characteristic Genetic O1981- of of (unit) Background 5 Corn plants Control plants Plant height B116 116.9 49 113.5 44 Mean (inches) 32.0- 36-72 26.7- 27-72 Range 136.5 138.5 0.7918 0.0067 0.0067 p-value CH01 110.4 48 106.8 46 Mean 32.7- 36-72 21.7- 31-72 Range 124.8 130.7 0.7632 0.01797 0.01797 p-value Grain weight B116 19.5 49 18.2 44 Mean (pounds per 3.8-27.0 36-72 4.0-37.4 27-72 Range plot) 0.2292 0.0067 0.0067 p-value CH01 19.9 48 18.8 46 Mean 6.0-30.6 36-72 2.0-31.1 31-72 Range 0.3662 0.01797 0.01797 p-value

TABLE-US-00013 TABLE 9 Agronomic performance results - reproductive parameters Genetic Agronomic Background Characteristic (same as in VCO-O1981-5 (unit) Table 8) Corn Control Days to 50% B116 72.6 73.3 Mean pollen shed 59-95 59-94 Range (# days) 0.6978 p-value CH01 72.3 72.9 Mean 57-93 56-94 Range 0.7365 p-value Days to 50% B116 74.6 75.0 Mean silking 59-97 59-95 Range (# days) 0.8087 p-value CH01 72.6 72.8 Mean 57-96 56-96 Range 0.9003 p-value Yield B116 143.0 130.7 Mean (bushel per 35.6-218.9 26.5-228.7 Range acre) 0.1584 p-value CH01 150.4 138.6 Mean 52.4-259.7 18.3-222.1 Range 0.1896 p-value

REFERENCES

Basra A., 1999. Heterosis and Hybrid Seed Production in Agronomic Crops (The Harwoth Press Inc.). Bernardo R., 2010. Breeding for quantitative traits in plants (2.sup.nd ed, Stemma press.com). Cheng, Z. M., Schnurr, J. A. and Kapaun, J. A., 1998. Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Reports. 646-649. De la Riva, G. A., Gonzalez-Cabrera, J., Vazquez-Padron, R., and Ayra-Pardo, C., 1998. Agrobacterium tumefaciens: A Natural Tool for Plant Transformation. Elec. J. of Biotech., 1, 118-133. Dellaporta S. L., Wood, J. and. Hicks, J. B., 1983. A plant DNA minipreparation: version II Plant Molecular Biology Reporter, 1, 19-21. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman, H. M. J., 1982. Molecular Applied Genetics, 1, 561-574. EFSA journal "Guidance for risk assessment of food and feed genetically modified plant, 2011; 9(5): 2150, p 10). Fang, L., Gross, P., Chen, C. and Lillis, M., 1992. Sequence of two acetohydroxyacid synthase genes from Zea mays, Plant Molecular Biology, 18, 1185-1187. Freeling M. and Walbot V., 1994. The Maize Handbook, Springer Lab Manuals. Gardner, R., Howarth, A., Hahn, P., Brown-Luedi, M., Shepherd, R., and Messing, J., 1981. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing, Nucleic Acids Research, 9, 2871-2888. Gelvin, S. B. 2005. Agricultural biotechnology: Gene exchange by Design. Nature 433, 583-584. Hallauer A. and Miranda J. B., 1988. Quantitative genetics in maize breeding. 2nd edition, Iowa State University press. Kelley P. M. and Tolan D. R., 1986. The complete amino acid sequence for the anaerobically induced aldolase from maize derived from cDNA clones. Plant Physiol. 82, 1076-1080. Komari, T., Hiei, Y., Saito, Y., Mural, N., and Kumashiro, T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165-174. Lawrence C. J., Dong Q., Polacco M. L., Seigfried T. E., Brendel V., 2004. Maize GDB, the community database for maize genetics and genomics. Nucleic Acids Res. 32. Database issue D393-D397. Otten, L., Salomone, J. Y., Helfer, A., Schmidt, J., Hammann, P. and De Ruffray, P. 1999 Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid, pTiC58 Plant Mol. Biol. 41 (6), 765-776. Pena L., 2005. Transgenic Plants: Methods and Protocols. Methods in Molecular Biology, Vol 286 Humana Press Inc. Sambrook, J., Fritsch, E. F., and Maniatis T., 1989. Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press.

SEQUENCE LISTINGS

1

23124DNAartificialminimal sequence junction in 5' 1ccaagaagat cagtactcaa acac 24225DNAartificialminimal sequence junction in 3' 2tttacaccgt tctcagaggg agatg 2535092DNAartificialtotal sequence of the T-DNA insert and the flanking genomic sequence 3tttcctcatt ttctttttcc cgcttttgtt tcaatttttc ttgggtaatg tacagtgagt 60atattttttc ttgttctttt tctcatggcc aaaatccaca atggatcgat gaattagctg 120tcgttgttgc caacaacaac aacagaacaa aatcacgtga cgtactagca caatgcaagt 180agccaaactg agcttccggg caccgacgaa cggttgcacg ccatcggcgg gaaggaacag 240gccgggctgt caatggacaa acgggccgcc aagctggagg gagtgtcatg ggctttgaga 300accatcgtca gggtccagtt tattcttttg tttttattaa aggcggtaaa ctcggggaac 360gaatatacta ggaaaaacac tagccagtca gagtcagtca aagtggactg agttaaaatt 420gcaacgacac acacgcagca gtcagggcgt cgggaatgaa caatggatga atttattata 480atctgaagaa aacgaaggga cacagccact acgaacactg gggagtgggg agtgaatgaa 540tgaatgcatt ccactggacc gttccagcgc ttcgtgtgcc tcgctagatg cgctgaacac 600tcgaacgcca tggacctcgc tccgctctct atatatagag ggaaggcctt cagtctactc 660ctcgggatat accactgaac gtcaccaaga agatcagtac tcaaacactg atagtttaaa 720ctgaagaagc ttaatttaaa tccctcgagt agcggccgct agcccgggca tagcttaatt 780cattatgtgg tctaggtagg ttctatatat aagaaaactt gaaatgttct aaaaaaaaat 840tcaagcccat gcatgattga agcaaacggt atagcaacgg tgttaacctg atctagtgat 900ctcttgcaat ccttaacggc cacctaccgc aggtagcaaa cggcgtcccc ctcctcgata 960tctccgcggc gacctctggc tttttccgcg gaattgcgcg gtggggacgg attccacgag 1020accgcgacgc aaccgcctct cgccgctggg ccccacaccg ctcggtgccg tagcctcacg 1080ggactctttc tccctcctcc cccgttataa attggcttca tcccctcctt gcctcatcca 1140tccaaatccc agtccccaat cccatccctt cgtcggagaa attcatcgaa gcgaagcgaa 1200tcctcgcgat cctctcaagg tactgcgagt tttcgatccc cctctcgacc cctcgtatgt 1260ttgtgtttgt cgtagcgttt gattaggtat gctttccctg tttgtgttcg tcgtagcgtt 1320tgattaggta tgctttccct gttcgtgttc atcgtagtgt ttgattaggt cgtgtgaggc 1380gatggcctgc tcgcgtcctt cgatctgtag tcgatttgcg ggtcgtggtg tagatctgcg 1440ggctgtgatg aagttatttg gtgtgatctg ctcgcctgat tctgcgggtt ggctcgagta 1500gatatgatgg ttggaccggt tggttcgttt accgcgctag ggttgggctg ggatgatgtt 1560gcatgcgccg ttgcgcgtga tcccgcagca ggacttgcgt ttgattgcca gatctcgtta 1620cgattatgtg atttggtttg gactttttag atctgtagct tctgcttatg tgccagatgc 1680gcctactgct catatgcctg atgataatca taaatggctg tggaactaac tagttgattg 1740cggagtcatg tatcagctac aggtgtaggg actagctaca ggtgtaggga cttgcgtcta 1800attgtttggt cctttactca tgttgcaatt atgcaattta gtttagattg tttgttccac 1860tcatctaggc tgtaaaaggg acactgctta gattgctgtt taatcttttt agtagattat 1920attatattgg taacttatta cccctattac atgccatacg tgacttctgc tcatgcctga 1980tgataatcat agatcactgt ggaattaatt agttgattgt tgaatcatgt ttcatgtaca 2040taccacggca caattgctta gttccttaac aaatgcaaat tttactgatc catgtatgat 2100ttgcgtggtt ctctaatgtg aaatactata gctacttgtt agtaagaatc aggttcgtat 2160gcttaatgct gtatgtgcct tctgctcatg cctgatgata atcatatatc actggaatta 2220attagttgat cgtttaatca tatatcaagt acataccatg gcacaatttt tagtcactta 2280acccatgcag attgaactgg tccctgcatg ttttgctaaa ttgttctatt ctgattagac 2340catatatcat gtattttttt ttggtaatgg ttctcttatt ttaaatgcta tatagttctg 2400gtacttgtta gaaagatctg cttcatagtt tagttgccta tccctcgaat taggatgctg 2460agcagctgat cctatagctt tgtttcatgt atcaattctt ttgtgttcaa cagtcagttt 2520ttgttagatt cattgtaact tatggtcgct tactcttctg gtcctcaatg cttgcagctg 2580cagaccatgg ccaccgccgc cgccgcgtct accgcgctca ctggcgccac taccgctgcg 2640cccaaggcga ggcgccgggc gcacctcctg gccacccgcc gcgccctcgc cgcgcccatc 2700aggtgctcag cggcgtcacc cgccatgccg atggctcccc cggccacccc gctccggccg 2760tggggcccca ccgatccccg caagggatcc ggcatggaaa ctgatcgcct tgtgatccca 2820ggatcgaaaa gcatcaccaa ccgggctttg cttttggctg ccgcagcgaa gggcacgtcg 2880gtcctggtga gaccattggt cagcgccgat acctcagcat tcaaaactgc aatccaggcc 2940ctcggtgcca acgtctcagc cgacggtgac gattgggtcg ttgaaggcct gggtcaggca 3000cccaacctcg acgccgacat ctggtgcgag gacgcaggta ctgtggcccg gttcctccct 3060ccattcgtag ccgcaggtca ggggaagttc accgtcgacg gatcagagca gctgcggcgg 3120cgcccgcttc ggcccgtggt cgacggcatc cgccacctgg gcgcccgcgt ctcctccgag 3180cagctgcccc ttacaattga agcgagcggg ctggcaggcg gggagtacga aattgaagcc 3240catcagagca gccagttcgc ctccggcctg atcatggccg ccccgtacgc gagacaaggc 3300ctgcgtgtga agataccaaa tcccgtgtca cagccctacc tcacgatgac actgcggatg 3360atgagggact tcggcattga gaccagcacc gacggagcca ccgtcagcgt ccctccaggg 3420cgctacacag cccggcggta tgaaatagaa ccggatgcgt caactgcgtc gtacttcgcc 3480gccgcttccg ccgtctctgg caggcgcttc gaatttcaag gccttggcac agacagcatc 3540caaggcgaca cgtcattctt caatgtactt gggcggctcg gtgcggaggt ccactgggca 3600tccaactcgg tcaccatacg gggaccggaa aggctgaccg gcgacattga agtggatatg 3660ggcgagattt cggacacctt catgacactc gcggcgattg cccctttggc cgatggaccc 3720atcacgataa ccaacattgg tcatgcacgg ttgaaggaat ccgaccgcat ctcagcgatg 3780gaaagcaacc tgcgcacgct cggtgtacaa accgacgtcg gacacgactg gatgagaatc 3840tacccctcta ccccgcacgg cggtagagtg aattgccacc gggaccacag gatcgctatg 3900gcgttttcaa tcctgggact gagagtggac gggattaccc tcgacgaccc tcaatgcgtc 3960gggaagacct ttcctggctt cttcgactac cttggacgcc ttttccccga aaaggcgctt 4020acgctccccg gctagggcgc gcctccttcg caagaccctt cctctatata aggaagttca 4080tttcatttgg agaggacacg ctgaaatcac cagtctctct ctacaaatct atctctctct 4140attttctcca taataatgtg tgagtagttc ccagataagg gaattagggt tcttataggg 4200tttcgctcac gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac 4260ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat ccactcgaga 4320cgcgtgaatt cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 4380tttgtttaca ccgttctcag agggagatgg gcggcaaggg cggcgggggt ggtggcaagg 4440gcggcggcgg gggtggtggc aagggcggag gaggttttgg tggcaagagc ggcggcgggg 4500gtggtggcaa gggcggagga ggtgttggtg gcaagagcgg cggcggcaag tcaggcggcg 4560gcggcggtgg gggctatggt ggtggaggga agtcaggctc cggcggcagt ggcggcgacg 4620gaatgatgaa ggcgcccggc ggcagtggcg agtacatctc ccgctctgtc ttcgaggcca 4680gcccgcaggt gttcttccat ggcctccacc agggaggagg caacaacaag tagatccatc 4740tagctagact gctgctgcta cttcacaagc ttgggacgat gtgtgatcat gcatgcttgg 4800actggcatca gtctctatgt agcttctgaa taaaataaaa tgtaacgatg ctcgattgtg 4860tttcacttgc tcgcttgttt cagccaagtt attatatatc atcaggctcg tacgtcagct 4920atatatatat atatatatat atatatatat atatatatat atatatatat atatatatat 4980atatatatat atatatatat atatatacac acacacacat atgcaggtgc atggattgtg 5040caacgcgaat gtgtgattgt gctaatccgt tagttgatgc cgtttgttgc tt 50924364DNASaccharum officinarum 4aattcattat gtggtctagg taggttctat atataagaaa acttgaaatg ttctaaaaaa 60aaattcaagc ccatgcatga ttgaagcaaa cggtatagca acggtgttaa cctgatctag 120tgatctcttg caatccttaa cggccaccta ccgcaggtag caaacggcgt ccccctcctc 180gatatctccg cggcgacctc tggctttttc cgcggaattg cgcggtgggg acggattcca 240cgagaccgcg acgcaaccgc ctctcgccgc tgggccccac accgctcggt gccgtagcct 300cacgggactc tttctccctc ctcccccgtt ataaattggc ttcatcccct ccttgcctca 360tcca 36451358DNASaccharum officinarum 5gtactgcgag ttttcgatcc ccctctcgac ccctcgtatg tttgtgtttg tcgtagcgtt 60tgattaggta tgctttccct gtttgtgttc gtcgtagcgt ttgattaggt atgctttccc 120tgttcgtgtt catcgtagtg tttgattagg tcgtgtgagg cgatggcctg ctcgcgtcct 180tcgatctgta gtcgatttgc gggtcgtggt gtagatctgc gggctgtgat gaagttattt 240ggtgtgatct gctcgcctga ttctgcgggt tggctcgagt agatatgatg gttggaccgg 300ttggttcgtt taccgcgcta gggttgggct gggatgatgt tgcatgcgcc gttgcgcgtg 360atcccgcagc aggacttgcg tttgattgcc agatctcgtt acgattatgt gatttggttt 420ggacttttta gatctgtagc ttctgcttat gtgccagatg cgcctactgc tcatatgcct 480gatgataatc ataaatggct gtggaactaa ctagttgatt gcggagtcat gtatcagcta 540caggtgtagg gactagctac aggtgtaggg acttgcgtct aattgtttgg tcctttactc 600atgttgcaat tatgcaattt agtttagatt gtttgttcca ctcatctagg ctgtaaaagg 660gacactgctt agattgctgt ttaatctttt tagtagatta tattatattg gtaacttatt 720acccctatta catgccatac gtgacttctg ctcatgcctg atgataatca tagatcactg 780tggaattaat tagttgattg ttgaatcatg tttcatgtac ataccacggc acaattgctt 840agttccttaa caaatgcaaa ttttactgat ccatgtatga tttgcgtggt tctctaatgt 900gaaatactat agctacttgt tagtaagaat caggttcgta tgcttaatgc tgtatgtgcc 960ttctgctcat gcctgatgat aatcatatat cactggaatt aattagttga tcgtttaatc 1020atatatcaag tacataccat ggcacaattt ttagtcactt aacccatgca gattgaactg 1080gtccctgcat gttttgctaa attgttctat tctgattaga ccatatatca tgtatttttt 1140tttggtaatg gttctcttat tttaaatgct atatagttct ggtacttgtt agaaagatct 1200gcttcatagt ttagttgcct atccctcgaa ttaggatgct gagcagctga tcctatagct 1260ttgtttcatg tatcaattct tttgtgttca acagtcagtt tttgttagat tcattgtaac 1320ttatggtcgc ttactcttct ggtcctcaat gcttgcag 13586198DNAZea mays 6atggccaccg ccgccgccgc gtctaccgcg ctcactggcg ccactaccgc tgcgcccaag 60gcgaggcgcc gggcgcacct cctggccacc cgccgcgccc tcgccgcgcc catcaggtgc 120tcagcggcgt cacccgccat gccgatggct cccccggcca ccccgctccg gccgtggggc 180cccaccgatc cccgcaag 19871242DNAArthrobacter globiformis 7atggaaactg atcgccttgt gatcccagga tcgaaaagca tcaccaaccg ggctttgctt 60ttggctgccg cagcgaaggg cacgtcggtc ctggtgagac cattggtcag cgccgatacc 120tcagcattca aaactgcaat ccaggccctc ggtgccaacg tctcagccga cggtgacgat 180tgggtcgttg aaggcctggg tcaggcaccc aacctcgacg ccgacatctg gtgcgaggac 240gcaggtactg tggcccggtt cctccctcca ttcgtagccg caggtcaggg gaagttcacc 300gtcgacggat cagagcagct gcggcggcgc ccgcttcggc ccgtggtcga cggcatccgc 360cacctgggcg cccgcgtctc ctccgagcag ctgcccctta caattgaagc gagcgggctg 420gcaggcgggg agtacgaaat tgaagcccat cagagcagcc agttcgcctc cggcctgatc 480atggccgccc cgtacgcgag acaaggcctg cgtgtgaaga taccaaatcc cgtgtcacag 540ccctacctca cgatgacact gcggatgatg agggacttcg gcattgagac cagcaccgac 600ggagccaccg tcagcgtccc tccagggcgc tacacagccc ggcggtatga aatagaaccg 660gatgcgtcaa ctgcgtcgta cttcgccgcc gcttccgccg tctctggcag gcgcttcgaa 720tttcaaggcc ttggcacaga cagcatccaa ggcgacacgt cattcttcaa tgtacttggg 780cggctcggtg cggaggtcca ctgggcatcc aactcggtca ccatacgggg accggaaagg 840ctgaccggcg acattgaagt ggatatgggc gagatttcgg acaccttcat gacactcgcg 900gcgattgccc ctttggccga tggacccatc acgataacca acattggtca tgcacggttg 960aaggaatccg accgcatctc agcgatggaa agcaacctgc gcacgctcgg tgtacaaacc 1020gacgtcggac acgactggat gagaatctac ccctctaccc cgcacggcgg tagagtgaat 1080tgccaccggg accacaggat cgctatggcg ttttcaatcc tgggactgag agtggacggg 1140attaccctcg acgaccctca atgcgtcggg aagacctttc ctggcttctt cgactacctt 1200ggacgccttt tccccgaaaa ggcgcttacg ctccccggct ag 12428270DNACauliflower mosaic virus 8tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga ggacacgctg 60aaatcaccag tctctctcta caaatctatc tctctctatt ttctccataa taatgtgtga 120gtagttccca gataagggaa ttagggttct tatagggttt cgctcacgtg ttgagcatat 180aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt 240cctaaaacca aaatccagta ctaaaatcca 2709700DNAartificialflanking 5' genomic sequence 9gttctcagag ggagatgggc ggcaagggcg gcgggggtgg tggcaagggc ggcggcgggg 60gtggtggcaa gggcggagga ggttttggtg gcaagagcgg cggcgggggt ggtggcaagg 120gcggaggagg tgttggtggc aagagcggcg gcggcaagtc aggcggcggc ggcggtgggg 180gctatggtgg tggagggaag tcaggctccg gcggcagtgg cggcgacgga atgatgaagg 240cgcccggcgg cagtggcgag tacatctccc gctctgtctt cgaggccagc ccgcaggtgt 300tcttccatgg cctccaccag ggaggaggca acaacaagta gatccatcta gctagactgc 360tgctgctact tcacaagctt gggacgatgt gtgatcatgc atgcttggac tggcatcagt 420ctctatgtag cttctgaata aaataaaatg taacgatgct cgattgtgtt tcacttgctc 480gcttgtttca gccaagttat tatatatcat caggctcgta cgtcagctat atatatatat 540atatatatat atatatatat atatatatat atatatatat atatatatat atatatatat 600atatatatat atatacacac acacacatat gcaggtgcat ggattgtgca acgcgaatgt 660gtgattgtgc taatccgtta gttgatgccg tttgttgctt 70010700DNAartificialflanking 5' genomic sequence 10atttcgagcg atttaagtat gcacagtata tagccttcat atgcatttta ataatttctg 60tcaattatct actacgggaa ataaaagtag aaaaataaag tccagaacta atgcatgaac 120atagcacatc aggtgtaaca agaattttac catattcaag catgtatttt tgcactaatt 180atttgcgaca ggaaataatt aatgaagata taaattgcga tagaaaaaca tgcttagttt 240tatttattat ttgcatcatt aatcatgaaa tatcatagaa ttaataatag ggagcatgat 300tataaattta tataaattca gcaggaattt tatttatata aaaaaacaag aataagatta 360gcaacttagt cgaattaaat caaaaaatgc taaggaggcg ccattatcct atgtgcataa 420gcacgctatg gatcccatga ccgtagcctt ttctgttgac cgcacatgca atatgaccat 480tgcatgcatg cacctcatgc actttgactt tgactggatc ttttcttacg ttggttggat 540gaggtcgctg cttatccgtg gcatgcagtg ccgcattcga agcgagcgga gggagagatt 600cggttttcgc tctctttccc gtatatcctt atcttcacga ctggttcaca tgcgtggccg 660gctctggcgt tccacaccag gcatcttggc gtaggactcc 7001122DNAartificialVCO-01981-5 primer F 11ccactgaacg tcaccaagaa ga 221220DNAartificialVCO-01981-5 primer R 12gccgctactc gagggattta 201320DNAartificialVCO-01981-5 probe 13cagtactcaa acactgatag 201417DNAartificialAldolase primer F 14agggaggacg cctccct 171522DNAartificialAldolase primer R 15accctgtacc agaagaccaa gg 221626DNAartificialAldolase probe 16tgaggacatc aacaaaaggc ttgcca 261727DNAartificialprimer Ace5-1 17acaggatcgc tatggcgttt tcaatcc 271828DNAartificialprimer Ace5-2 18atgcgtcggg aagacctttc ctggcttc 281927DNAartificialprimer O39 19caccagggag gaggcaacaa caagtag 272027DNAartificialprimer Scubi-NewR 20agaaagagtc ccgtgaggct acggcac 272128DNAartificialprimer Scubi2-Rev 21ctgggatttg gatggatgag gcaaggag 282227DNAartificialScubi1-Rev 22agaggtcgcc gcggagatat cgaggag 2723413PRTartificialprotein sequence GRG23ACE5 23Met Glu Thr Asp Arg Leu Val Ile Pro Gly Ser Lys Ser Ile Thr Asn 1 5 10 15 Arg Ala Leu Leu Leu Ala Ala Ala Ala Lys Gly Thr Ser Val Leu Val 20 25 30 Arg Pro Leu Val Ser Ala Asp Thr Ser Ala Phe Lys Thr Ala Ile Gln 35 40 45 Ala Leu Gly Ala Asn Val Ser Ala Asp Gly Asp Asp Trp Val Val Glu 50 55 60 Gly Leu Gly Gln Ala Pro Asn Leu Asp Ala Asp Ile Trp Cys Glu Asp 65 70 75 80 Ala Gly Thr Val Ala Arg Phe Leu Pro Pro Phe Val Ala Ala Gly Gln 85 90 95 Gly Lys Phe Thr Val Asp Gly Ser Glu Gln Leu Arg Arg Arg Pro Leu 100 105 110 Arg Pro Val Val Asp Gly Ile Arg His Leu Gly Ala Arg Val Ser Ser 115 120 125 Glu Gln Leu Pro Leu Thr Ile Glu Ala Ser Gly Leu Ala Gly Gly Glu 130 135 140 Tyr Glu Ile Glu Ala His Gln Ser Ser Gln Phe Ala Ser Gly Leu Ile 145 150 155 160 Met Ala Ala Pro Tyr Ala Arg Gln Gly Leu Arg Val Lys Ile Pro Asn 165 170 175 Pro Val Ser Gln Pro Tyr Leu Thr Met Thr Leu Arg Met Met Arg Asp 180 185 190 Phe Gly Ile Glu Thr Ser Thr Asp Gly Ala Thr Val Ser Val Pro Pro 195 200 205 Gly Arg Tyr Thr Ala Arg Arg Tyr Glu Ile Glu Pro Asp Ala Ser Thr 210 215 220 Ala Ser Tyr Phe Ala Ala Ala Ser Ala Val Ser Gly Arg Arg Phe Glu 225 230 235 240 Phe Gln Gly Leu Gly Thr Asp Ser Ile Gln Gly Asp Thr Ser Phe Phe 245 250 255 Asn Val Leu Gly Arg Leu Gly Ala Glu Val His Trp Ala Ser Asn Ser 260 265 270 Val Thr Ile Arg Gly Pro Glu Arg Leu Thr Gly Asp Ile Glu Val Asp 275 280 285 Met Gly Glu Ile Ser Asp Thr Phe Met Thr Leu Ala Ala Ile Ala Pro 290 295 300 Leu Ala Asp Gly Pro Ile Thr Ile Thr Asn Ile Gly His Ala Arg Leu 305 310 315 320 Lys Glu Ser Asp Arg Ile Ser Ala Met Glu Ser Asn Leu Arg Thr Leu 325 330 335 Gly Val Gln Thr Asp Val Gly His Asp Trp Met Arg Ile Tyr Pro Ser 340 345 350 Thr Pro His Gly Gly Arg Val Asn Cys His Arg Asp His Arg Ile Ala 355 360 365 Met Ala Phe Ser Ile Leu Gly Leu Arg Val Asp Gly Ile Thr Leu Asp 370 375 380 Asp Pro Gln Cys Val Gly Lys Thr Phe Pro Gly Phe Phe Asp Tyr Leu 385 390 395 400 Gly Arg Leu Phe Pro Glu Lys Ala Leu Thr Leu Pro Gly 405 410

Claims

The invention claimed is:

1. A glyphosate tolerant maize plant comprising in its genome the nucleotide sequence as set forth in SEQ ID NO: 3.

2. The glyphosate tolerant maize plant of claim 1, wherein the glyphosate tolerant maize plant is obtained by breeding a maize plant with a maize plant grown from seeds deposited with NCIMB with accession number 41842.

3. The glyphosate tolerant maize plant of claim 2, wherein the glyphosate tolerant maize plant is an hybrid maize plant.

4. The glyphosate tolerant maize plant of claim 1, wherein a part of the glyphosate tolerant maize plant, cells or seeds comprise a nucleotide sequence as set forth in SEQ ID NO: 3.